A systematic investigation of protein phosphorylation in the rat central nervous system has revealed that a family of substrates for cAMP-dependent protein kinase, termed DARPPs (for dopamine and cAMP-regulated phosphoproteins), are highly enriched in athe basal ganglia. In this brain region, dopamine, through binding to D1 receptors, increases cAMP levels leading to activation of PKA and phosphorylation of this family of substrates. Studies over the last few years now indicate that members of the DARPP family mediate many of the actions of dopamine in this brain region. There is also considerable evidence to indicate that the acute and chronic actions of psychomotor stimulants (e.g. cocaine and amphetamine) are mediated through augmentation of neurotransmission in mesolimbic and nigrostriatal dopamine systems. The major aim of the present proposal will be to study the role of two of the DARPP family, namely DARPP-21 and DARPP- 16, in the actions of these psychomotor stimulants. Specific Aims will be: I. to use gene-targeting techniques to "knock-out" the genes for DARPP-21 and DARPP-16 in mice. This will provide information in an intact animal model concerning the physiological role these proteins play in dopaminergic neurotransmission and ina the actions of psychomotor stimulants; II. to identify the function of DARPP-21 and DARPP-16 through the use of the yeast two-hybrid method and other affinity methods to identify the targets for each of these two proteins. These studies will include characterization of each DARPP-21 and DARPP-16 binding protein using biochemical, immunological and structural methods, including cloning of cDNAs for each protein, and examination of how phosphorylation of each DARPP influences its interaction with identified binding proteins. The role played by intracellular targets for DARPP-21 and DARPP-16 in the actions of cocaine and amphetamines will also be studied; III. to purify and characterize other novel DARPPs that are localized in the neostriatum and nucleus accumbens. The characterization of basal ganglia phosphoproteins, within specific neurons which are affected by psychostimulants, will provide a rational new approach to developing drugs that specifically affect these phosphoproteins or their targets. Thus, these studies may have potential implication int he development of new therapeutic approaches to the treatment of such drugs of abuse.